Elkady Ayman I, Hussein Rania A, El-Assouli Sufian M
Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia E-mail :
Asian Pac J Cancer Prev. 2015;16(17):7943-57. doi: 10.7314/apjcp.2015.16.17.7943.
Colorectal cancer is one of the leading causes of death in the world. The aim of this study was to investigate the growth-suppression potentiality of a crude saponin extract (CSENS) prepared from medicinal herb, Nigella sativa, on human colon cancer cells, HCT116.
HCT116 cells were subjected to increasing doses of CSENS for 24, 48 and 72 h, and then harvested and assayed for cell viability by WST-1. Flow cytometry analyses, cell death detection ELISA, fluorescent stains (Hoechst 33342 and acridine orange/ethidium bromide), DNA laddering and comet assays were carried out to confirm the apoptogenic effects of CSENS. Luciferase reporter gene assays, quantitative reverse transcription-polymerase chain reaction and Western blot analyses were performed to assess the impact of CAERS and CFEZO on the expression levels of key regulatory proteins in HCT116 cells.
The results demonstrated that CSENS inhibited proliferation and induced apoptosis. Apoptosis was confirmed by flow cytometry analyses, while CSENS-treated cells exhibited morphological hallmarks of apoptosis including cell shrinkage, irregularity in cellular shape, cellular detachment and chromatin condensation. Biochemical signs of apoptosis, such as DNA degradation, were observed by comet assay and gel electrophoresis. The pro-apoptotic effect of CSENS was caspase-3-independent and associated with increase of the Bax/Bcl-2 ratio. CSENS treatment down-regulated transcriptional and DNA-binding activities of NF-κB and AP-1 proteins, associated with down-regulation of their target oncogenes, c-Myc, cyclin D1 and survivin. On the other hand, CSENS up-regulated transcriptional and DNA-binding activities of Nrf2 and expression of cytoprotective genes. In addition, CSENS modulated the expression levels of ERK1/2 MAPK, p53 and p21.
These findings suggest that CSENS may be a valuable agent for treatment of colon cancer.
结直肠癌是全球主要的死亡原因之一。本研究旨在探讨从药用植物黑种草中制备的粗皂苷提取物（CSENS）对人结肠癌细胞HCT116的生长抑制潜力。
用递增剂量的CSENS处理HCT116细胞24、48和72小时，然后收获细胞，并用WST-1检测细胞活力。进行流式细胞术分析、细胞死亡检测ELISA、荧光染色（Hoechst 33342和吖啶橙/溴化乙锭）、DNA梯状条带分析和彗星试验，以证实CSENS的凋亡诱导作用。进行荧光素酶报告基因试验、定量逆转录-聚合酶链反应和蛋白质印迹分析，以评估CSENS对HCT116细胞中关键调节蛋白表达水平的影响。
结果表明CSENS抑制增殖并诱导凋亡。流式细胞术分析证实了凋亡，而经CSENS处理的细胞表现出凋亡的形态学特征，包括细胞收缩、细胞形状不规则、细胞脱离和染色质浓缩。彗星试验和凝胶电泳观察到凋亡的生化迹象，如DNA降解。CSENS的促凋亡作用不依赖于半胱天冬酶-3，且与Bax/Bcl-2比值增加有关。CSENS处理下调了NF-κB和AP-1蛋白的转录和DNA结合活性，这与它们的靶癌基因c-Myc、细胞周期蛋白D1和存活素的下调有关。另一方面，CSENS上调了Nrf2的转录和DNA结合活性以及细胞保护基因的表达。此外，CSENS调节了ERK1/2 MAPK、p53和p21的表达水平。
这些发现表明CSENS可能是一种治疗结肠癌的有价值的药物。
